Effect of abrupt weaning at housing on leukocyte distribution, functional activity of neutrophils, and acute phase protein response of beef calves.
Sixteen, spring-born, single suckled, castrated male calves of Limousin x Holstein-Friesian and Simmental x Holstein-Friesian dams respectively, were used to investigate the effect of weaning on total leukocyte and differential counts, neutrophil functional activity, lymphocyte immunophenotypes, and acute phase protein response. Calves grazed with their dams until the end of the grazing season when they were housed in a slatted floor shed. On the day of housing, calves were assigned to a treatment, (i) abruptly weaned (W: n = 8) or (ii) non-weaned (controls) (C: n = 8). Weaned calves were housed in pens without their dams, whereas non-weaned (control) calves were housed with their dams. Blood was collected on day -7, 0 (housing), 2, 7, and 14 to determine total leukocyte and differential counts and concentration of fibrinogen and haptoglobin. Lymphocyte immunophenotypes were characterised using selected surface antigens (CD4+, CD8+, WC1+ (gammadelta T cells), MHC Class II+ lymphocytes), and the functional activities of neutrophils (surface expression of L-selectin (CD62L), phagocytic and oxidative burst activity) were investigated using flow cytometry. Treatment x sampling time interactions (P < 0.05) were detected for total leukocyte and neutrophil counts, all lymphocyte subsets, mean fluorescence intensity of CD62L+ neutrophils, and percentage neutrophils performing phagocytosis. On d 2, total leukocyte and neutrophil count increased (P < 0.001), and percentage CD4+ and CD8+ lymphocytes, percentage phagocytic neutrophils, mean fluorescence intensity of CD62L+ neutrophils decreased (P < 0.05) in W compared with baseline (d 0), whereas they were unchanged (P > 0.05) in C. On d 2, percentage WC1+ lymphocytes decreased (P < 0.05), whereas percentage MHC class II+ lymphocytes increased (P < 0.05) in W and C, however the magnitude of change was greater in W than C. There were no treatment x sampling time interactions (P > 0.05) for monocyte, eosinophil, and basophil counts, percentage G1+ neutrophils, or percentage oxidative burst positive neutrophils. Abrupt weaning resulted in increased neutrophil counts and impaired trafficking and phagocytic function. Together with the changes in lymphocyte subsets, the results suggest that there was a greater transitory reduction in immune function at housing in abruptly weaned than non-weaned beef calves.


Family Socioeconomic Status Moderates Associations Between Television Viewing and School Readiness Skills.
We examined whether the negative relation between television viewing that exceeds the recommendations of the American Academy of Pediatrics (AAP) and school readiness varied by family income. Data were collected from 807 children from diverse backgrounds. Parents reported hours of television viewing, as well as family income. Children were assessed using measures of math, knowledge of letters and words, and executive function (EF). Television viewing was negatively associated with math and EF but not with letter and word knowledge. An interaction between television viewing and family income indicated that the effect of television viewing in excess of the AAP recommended maximum had negative associations with math and EF that increased as a linear function of family income. Furthermore, EF partially mediated the relation between television viewing and math. Television viewing is negatively associated with children's school readiness skills, and this association increased as family income decreased. Active efforts to reinforce AAP guidelines to limit the amount of television children watch should be made, especially for children from middle- to lower-income families.


Good Cascade Impactor Practice (GCIP) and considerations for "in-use" specifications.
The multi-stage cascade impactor (CI) is widely used to determine aerodynamic particle size distributions (APSDs) of orally inhaled products. Its size-fractionating capability depends primarily on the size of nozzles of each stage. Good Cascade Impactor Practice (GCIP) requires that these critical dimensions are linked to the accuracy of the APSD measurement based on the aerodynamic diameter size scale. Effective diameter (Deff) is the critical dimension describing any nozzle array, as it is directly related to stage cut-point size (d50). d50 can in turn be determined by calibration using particles of known aerodynamic diameter, providing traceability to the international length standard. Movements in Deff within manufacturer tolerances for compendial CIs result in the worst case in shifts in d50 of <±10%. Stage mensuration therefore provides satisfactory control of measurement accuracy. The accurate relationship of Deff to d50 requires the CI system to be leak-free, which can be checked by sealing the apparatus at the entry to the induction port and isolating it from the vacuum source and measuring the rate of pressure rise before each use. Mensuration takes place on an infrequent basis compared with the typical interval between individual APSD determinations. Measurement of stage flow resistance (pressure drop; ΔPstage) could enable the user to know that the CI stages are fit for use before every APSD measurement, by yielding an accurate measure of Deff. However, more data are needed to assess the effects of wear and blockage before this approach can be advocated as part of GCIP.


Microscopy of biological sample through advanced diffractive optics from visible to X-ray wavelength regime.
The aim of this report is to demonstrate a unified version of microscopy through the use of advanced diffractive optics. The unified scheme derives from the technical possibility of realizing front wave engineering in a wide range of electromagnetic spectrum. The unified treatment is realized through the design and nanofabrication of phase diffractive elements (PDE) through which wave front beam shaping is obtained. In particular, we will show applications, by using biological samples, ranging from micromanipulation using optical tweezers to X-ray differential interference contrast (DIC) microscopy combined with X-ray fluorescence. We report some details on the design and physical implementation of diffractive elements that besides focusing also perform other optical functions: beam splitting, beam intensity, and phase redistribution or mode conversion. Laser beam splitting is used for multiple trapping and independent manipulation of micro-beads surrounding a cell as an array of tweezers and for arraying and sorting microscopic size biological samples. Another application is the Gauss to Laguerre-Gauss mode conversion, which allows for trapping and transfering orbital angular momentum of light to micro-particles immersed in a fluid. These experiments are performed in an inverted optical microscope coupled with an infrared laser beam and a spatial light modulator for diffractive optics implementation. High-resolution optics, fabricated by means of e-beam lithography, are demonstrated to control the intensity and the phase of the sheared beams in x-ray DIC microscopy. DIC experiments with phase objects reveal a dramatic increase in image contrast compared to bright-field x-ray microscopy. Besides the topographic information, fluorescence allows detection of certain chemical elements (Cl, P, Sc, K) in the same setup, by changing the photon energy of the x-ray beam.


Parafibromin/Hyrax activates Wnt/Wg target gene transcription by direct association with beta-catenin/Armadillo.
The Wnt pathway controls cell fates, tissue homeostasis, and cancer. Its activation entails the association of beta-catenin with nuclear TCF/LEF proteins and results in transcriptional activation of target genes. The mechanism by which nuclear beta-catenin controls transcription is largely unknown. Here we genetically identify a novel Wnt/Wg pathway component that mediates the transcriptional outputs of beta-catenin/Armadillo. We show that Drosophila Hyrax and its human ortholog, Parafibromin, components of the Polymerase-Associated Factor 1 (PAF1) complex, are required for nuclear transduction of the Wnt/Wg signal and bind directly to the C-terminal region of beta-catenin/Armadillo. Moreover, we find that the transactivation potential of Parafibromin/Hyrax depends on the recruitment of Pygopus to beta-catenin/Armadillo. Our results assign to the tumor suppressor Parafibromin an unexpected role in Wnt signaling and provide a molecular mechanism for Wnt target gene control, in which the nuclear Wnt signaling complex directly engages the PAF1 complex, thereby controlling transcriptional initiation and elongation by RNA Polymerase II.


The single-cell eQTLGen consortium.
In recent years, functional genomics approaches combining genetic information with bulk RNA-sequencing data have identified the downstream expression effects of disease-associated genetic risk factors through so-called expression quantitative trait locus (eQTL) analysis. Single-cell RNA-sequencing creates enormous opportunities for mapping eQTLs across different cell types and in dynamic processes, many of which are obscured when using bulk methods. Rapid increase in throughput and reduction in cost per cell now allow this technology to be applied to large-scale population genetics studies. To fully leverage these emerging data resources, we have founded the single-cell eQTLGen consortium (sc-eQTLGen), aimed at pinpointing the cellular contexts in which disease-causing genetic variants affect gene expression. Here, we outline the goals, approach and potential utility of the sc-eQTLGen consortium. We also provide a set of study design considerations for future single-cell eQTL studies.


Long-term occurrence of death and cardiovascular events in patients with transient ischaemic attack or minor ischaemic stroke: comparison between arterial and cardiac source of the index event.
Published data suggest that patients with cerebral ischaemia and atrial fibrillation (CIAF) have higher inhospital mortality than patients with cerebral ischaemia of arterial origin (CIAO). Data on long term risks are scarce. We compared the long term risks of death and vascular events (VE) between these groups. We extended the follow-up of 2473 patients from the Dutch TIA Trial (recruitment March 1986 to March 1989, all treated with aspirin; CIAO) and 186 Dutch participants of the European Atrial Fibrillation Trial (recruitment June 1988 to May 1992, 26% on anticoagulants during the trial; CIAF). Hazard ratios (HRs) for death and VE of CIAF versus CIAO were analysed by means of Cox regression analysis and adjusted for age, sex and several cardiovascular risk factors. After a mean follow-up of 10.1 years, 1484 patients with CIAO had died and 1336 had suffered at least one VE (377 cardiac, 455 stroke). Mean follow-up of the CIAF patients was 6.8 years; 150 patients had died and 136 had suffered at least one VE (41 cardiac, 63 stroke). Adjusted HRs (CIAF vs CIAO) were 1.46 (95% CI 1.22 to 1.74) for death, 1.49 (1.24 to 1.79) for first VE, 1.94 (1.47 to 2.55) for first stroke and 1.41 (1.01 to 1.96) for first cardiac event. These HRs were essentially the same as those for the duration of the trials. Our study shows that the long term risk of death or vascular events is 1.5 times higher in patients with CIAF than in those with CIAO, after adjustment for differences between the groups.


Regional Anesthesia Associated With Decreased Inpatient and Outpatient Opioid Demand in Tibial Plateau Fracture Surgery.
Regional anesthesia (RA) has been used to reduce pain and opioid usage in elective orthopedic surgery. The hypothesis of this study was that RA would be associated with decreased opioid demand in tibial plateau fracture surgery. Inpatient opioid consumption and 90-day outpatient opioid prescribing in all patients ≥18 years of age undergoing tibial plateau fracture surgery from July 2013 to July 2018 (n = 264) at a single, level I trauma center were recorded. The presence or absence of perioperative RA was noted. Of 60 patients receiving RA, 52 underwent peripheral nerve blockade (PNB) with single-shot sciatic-popliteal (40.0%; n = 24), femoral (26.7%; n = 16), adductor canal (18.3%; n = 11), or fascia iliaca (1.7%; n = 1) block with ropivacaine. Ten patients received epidural analgesia (EA) with either single-shot spinal (11.7%; n = 7) blocks or continuous epidural (5.0%; n = 3). Additional baseline and treatment characteristics were recorded, including age, sex, race, body mass index (BMI), smoking, chronic opioid use, American Society of Anesthesiologists (ASA) score, injury mechanism, additional injuries, open injury, and additional inpatient surgery. Statistical models, including multivariable generalized linear models with propensity score weighting to adjust for baseline patient and treatment characteristics, were used to assess perioperative opioid demand with and without RA. RA was associated with reduced inpatient opioid usage from 0 to 24 hours postoperatively of approximately 5.2 oxycodone 5-mg equivalents (0.74 incident rate ratio [IRR]; 0.63-0.86 CI; P < .001) and from 24 to 48 hours postoperatively of approximately 2.9 oxycodone 5-mg equivalents (0.78 IRR; 0.64-0.95 CI; P = .014) but not at 48 to 72 hours postoperatively. From 1 month preoperatively to 2 weeks postoperatively, RA was associated with reduced outpatient opioid prescribing of approximately 24.0 oxycodone 5-mg equivalents (0.87; 0.75-0.99; P = .044) and from 1 month preoperatively to 90 days postoperatively of approximately 44.0 oxycodone 5-mg equivalents (0.83; 0.71-0.96; P = .011), although there was no significant difference from 1 month preoperatively to 6 weeks postoperatively. There were no cases of acute compartment syndrome in this cohort. In tibial plateau fracture surgery, RA was associated with reduced inpatient opioid consumption up to 48 hours postoperatively and reduced outpatient opioid demand up to 90 days postoperatively without an associated risk of acute compartment syndrome. RA should be considered for patients undergoing tibial plateau fracture fixation.


Improvement of the clinical applicability of the Genomic Grade Index through a qRT-PCR test performed on frozen and formalin-fixed paraffin-embedded tissues.
Proliferation and tumor differentiation captured by the genomic grade index (GGI) are important prognostic indicators in breast cancer (BC) especially for the estrogen receptor positive (ER+) disease. The aims of this study were to convert this microarray index to a qRT-PCR assay (PCR-GGI), which could be realized on formalin fixed paraffin embedded samples (FFPE), and to assess its prognostic performance and predictive value of clinical benefit in early and advanced ER+ BC patients treated with tamoxifen. The accuracy and concordance of the PCR-GGI with the GGI was assessed using BC patients for which frozen and FFPE tissues as well as microarray data were available (n = 19). The evaluation of the prognostic value of the PCR-GGI was assessed on FFPE material using a consecutive series of 212 systemically treated early BC patients. The predictive performance for tamoxifen benefit was assessed using two ER+ BC populations treated either with adjuvant tamoxifen only (n = 77+139) or first-line tamoxifen for advanced disease (n = 270). The PCR-GGI is based on the expression of 8 genes (4 representative of the GGI and 4 reference genes). A significant correlation was observed between the microarray-derived GGI and the qRT-PCR assay using frozen (rho = 0.95, p < 10E-06) and FFPE material (rho = 0.89, p < 10E-06). The prognostic performance of the PCR-GGI was confirmed on FFPE samples (HRunivar. = 1.89; [95CI:1.01-3.54], p = 0.05). The PCR-GGI further identified two subgroups of patients with statistically different time to distant metastasis free survival (DMFS) across the two cohorts of ER+ BC patients treated with adjuvant tamoxifen. Additionally, the PCR-GGI was associated with response to tamoxifen in the advanced setting (HRunivar. = 1.98; [95CI:1.51-2.59], p = 6.9E-07). PCR-GGI recapitulates in an accurate and reproducible manner the performances of the GGI using frozen and FFPE samples.


Role of PPARα and Its Agonist in Renal Diseases.
Peroxisome proliferator-activated receptor (PPAR)-α, a member of a large nuclear receptor superfamily, plays a major role in the regulation of lipid metabolism. Recently, PPARα activation has been shown to confer additional benefits on endothelial function, kidney function, and anti-inflammation, suggesting that PPARα agonists may be good candidates for treating acute renal failure. In clinical application, PPAR-α activators, such as hypolipidemic drugs in fibric acid class, were proven to have therapeutic effects on metabolic syndrome and cardiovascular disease. This paper focuses on signaling pathways, ligand selectivity, and physio-pathological roles of PPARα in kidney diseases and the therapeutic utility of PPARα modulators in the treatment of diabetes and inflammation-induced nephropathy. Implication of new and more potent PPAR-α activators could provide important insights into the overall benefits of activating PPAR-α clinically for the treatment of dyslipidemia and the prevention of diabetic or inflammation-induced nephropathy in the future.


HPV testing in first-void urine provides sensitivity for CIN2+ detection comparable with a smear taken by a clinician or a brush-based self-sample: cross-sectional data from a triage population.
To compare the sensitivity of high-risk human papillomavirus (hrHPV) and genotype detection in self-collected urine samples in the morning (U1), and later on (U2), brush-based self-samples (SS), and clinician-taken smears (CTS) for detecting cervical intraepithelial neoplasia grade 2+ (CIN2+) in a colposcopic referral population. Cross-sectional single-centre study. A colposcopy clinic in Spain. A cohort of 113 women referred for colposcopy after an abnormal Pap smear. Women undergoing colposcopy with biopsy for abnormal Pap smears were sent a device (Colli-Pee™, Novosanis, Wijnegem, Belgium) to collect U1 on the morning of colposcopy. U2, CTS, and SS (Evalyn brush™, Rovers Medical Devices B.V., Oss, the Netherlands) were also analysed. All samples were tested for HPV DNA using the analytically sensitive SPF10-DEIA-LiPA25 assay and the clinically validated GP5+/6+-EIA-LMNX. Histologically confirmed CIN2+ and hrHPV positivity for 14 high-risk HPV types. Samples from 91 patients were analysed. All CIN3 cases (n = 6) tested positive for hrHPV in CTS, SS, U1, and U2 with both HPV assays. Sensitivity for CIN2+ with the SPF10 system was 100, 100, 95, and 100%, respectively. With the GP5+/6+ assay, sensitivity was 95% in all sample types. The sensitivities and specificities for both tests on each of the sample types did not significantly differ. There was 10-14% discordance on hrHPV genotype. CIN2+ detection using HPV testing of U1 shows a sensitivity similar to that of CTS or brush-based SS, and is convenient. There was substantial to almost excellent agreement between all samples on genotype with both hrHPV assays. There was no advantage in testing U1 compared with U2 samples. Similar CIN2+ sensitivity for HPV testing in first-void urine, physician-taken smear and brush-based self-sample.


Alterations in renal mitochondrial respiration in response to the reactive oxoaldehyde methylglyoxal.
Chronic hyperglycemia has been linked to alterations in mitochondrial function, suggesting an important role in the pathophysiology of the complications of diabetes mellitus. In the diabetic kidney, ultrastructural changes in mitochondria are associated with impaired tubular function. The goal of this study was to determine if methylglyoxal (MGO), a dicarbonyl compound reaching high levels in hyperglycemic conditions, has direct toxicity for renal mitochondria. Intact mitochondria isolated from the renal cortex of rats were incubated with MGO to determine 1) its effect on mitochondrial respiration, 2) the conditions under which MGO exerts these effects, and 3) the potential mitochondrial targets of MGO influence. This study demonstrates that MGO has an inhibitory effect on both the tricarboxylic acid cycle and the electron respiratory chain. The modifications appear to be specific to certain mitochondrial proteins. Alterations of these proteins lead to disturbances in mitochondria that may play an important role in renal cellular toxicity and in the development of diabetic nephropathy.


Increase in CFC-11 emissions from eastern China based on atmospheric observations.
The recovery of the stratospheric ozone layer relies on the continued decline in the atmospheric concentrations of ozone-depleting gases such as chlorofluorocarbons1. The atmospheric concentration of trichlorofluoromethane (CFC-11), the second-most abundant chlorofluorocarbon, has declined substantially since the mid-1990s2. A recently reported slowdown in the decline of the atmospheric concentration of CFC-11 after 2012, however, suggests that global emissions have increased3,4. A concurrent increase in CFC-11 emissions from eastern Asia contributes to the global emission increase, but the location and magnitude of this regional source are unknown3. Here, using high-frequency atmospheric observations from Gosan, South Korea, and Hateruma, Japan, together with global monitoring data and atmospheric chemical transport model simulations, we investigate regional CFC-11 emissions from eastern Asia. We show that emissions from eastern mainland China are 7.0 ± 3.0 (±1 standard deviation) gigagrams per year higher in 2014-2017 than in 2008-2012, and that the increase in emissions arises primarily around the northeastern provinces of Shandong and Hebei. This increase accounts for a substantial fraction (at least 40 to 60 per cent) of the global rise in CFC-11 emissions. We find no evidence for a significant increase in CFC-11 emissions from any other eastern Asian countries or other regions of the world where there are available data for the detection of regional emissions. The attribution of any remaining fraction of the global CFC-11 emission rise to other regions is limited by the sparsity of long-term measurements of sufficient frequency near potentially emissive regions. Several considerations suggest that the increase in CFC-11 emissions from eastern mainland China is likely to be the result of new production and use, which is inconsistent with the Montreal Protocol agreement to phase out global chlorofluorocarbon production by 2010.


Interventions for preventing back pain among office workers - a systematic review and network meta-analysis.
Back pain is common in the working population. This systematic review with network meta-analysis (NMA) aimed to compare the effects of interventions for preventing back pain among office workers. We searched eight databases and additional sources up to March 2021. We included randomized controlled trials (RCT) and cluster RCT focusing on office workers, comparing work-related interventions aimed at preventing back pain (defined as pain in any part of the spine) to a control condition and assessing back pain and/or work absence. Further outcomes considered were adverse events and participants' satisfaction. We performed both frequentist and component NMA. Risk of bias (RoB) was evaluated using RoB 2 and certainty of the evidence (CoE) was assessed using GRADE. We screened 9809 records and included 24 studies with a total of 7080 participants. RoB was assessed as "some concerns" or "high" for all studies and outcomes. Included studies investigated multicomponent interventions, ergonomics, physical activity, education, behavioral interventions and no/minimal interventions. Effects were mostly not statistically significant and based on low/very low CoE. Physical activity probably reduces days of work absence slightly [mean difference (MD) -1.10, 95% confidence interval (CI) -2.07- -0.13], and combining physical activity and ergonomics may reduce back pain intensity (standardized MD -0.41, 95% CI -0.80- -0.02) when compared to no/minimal intervention. A large proportion of participants were satisfied with the interventions, adverse events were rarely assessed. We observed mostly minor effects of interventions on back pain and work absence among office workers. The practical relevance of these effects is questionable.


Relative abundance of selenoprotein P isoforms in human plasma depends on genotype, se intake, and cancer status.
Selenium (Se), a dietary trace metal essential for human health, is incorporated into selenoproteins as selenocysteine. Selenoprotein P (SePP), the major plasma selenoprotein, has both transport and antioxidant functions. In humans, it exists in plasma as two isoforms of approximately 50 and 60 kDa. This study investigated the effect of polymorphisms in the SEPP-1 gene, Se supplementation, and disease status on the proportions of SePP plasma isoforms. SePP was isolated from plasma from healthy volunteers, before and after a 6-week supplementation with 100 microg sodium selenite, and from colon cancer patients and controls. SePP isoform distribution was analysed by Western blot. In healthy volunteers, the relative abundance of each isoform depended on two SEPP-1 polymorphisms: rs3877899, predicted to cause an Ala-to-Thr amino acid change at position 234, and rs7579, located in the 3'-untranslated region of SEPP-1 mRNA. The difference between genotypes disappeared after Se supplementation. A genotype-dependent reduction was seen in the proportion of the 60-kDa isoform in patients with colorectal cancer compared with controls. We conclude that functional polymorphisms in the SEPP-1 gene influence the proportion of SePP isoforms in plasma. An elevated proportion of the 60-kDa isoform of SePP may increase selenoprotein synthesis and reduce colorectal cancer risk.


Modulatory effect of acetyl-L-carnitine on amyloid precursor protein metabolism in hippocampal neurons.
Alzheimer Disease is the most common chronic neurodegenerative disorder associated with aging. Nevertheless, its pharmacological therapy is still an unresolved issue. In double-blind controlled studies, acetyl-L-carnitine (ALC) demonstrated beneficial effects on Alzheimer's disease. However, the mechanisms behind its neuroprotective ability remain to be fully established. In this study, the effect of acetyl-L-carnitine on amyloid precursor protein (APP) metabolism was investigated by in vitro models, both in a neuroblastoma cell line and in primary hippocampal cultures. We found that ALC treatment stimulates alpha-secretase activity and physiological APP metabolism. In particular, ALC favors the delivery of ADAM10 (a disintegrin and metalloproteinase 10, the most accredited candidate for alpha-secretase) to the post-synaptic compartment, and consequently positively modulates its enzymatic activity towards APP. Our findings suggest that the benefits of ALC reported in previous clinical studies are underscored by the specific biological mechanism of this compound on APP metabolism. In fact, ALC can directly influence the primary event in Alzheimer's disease pathogenesis, i.e. the Amyloid beta cascade, promoting alpha-secretase activity and directly affecting the release of the non amyloidogenic metabolite.


Synthetic enzyme mixtures for biomass deconstruction: production and optimization of a core set.
The high cost of enzymes is a major bottleneck preventing the development of an economically viable lignocellulosic ethanol industry. Commercial enzyme cocktails for the conversion of plant biomass to fermentable sugars are complex mixtures containing more than 80 proteins of suboptimal activities and relative proportions. As a step toward the development of a more efficient enzyme cocktail for biomass conversion, we have developed a platform, called GENPLAT, that uses robotic liquid handling and statistically valid experimental design to analyze synthetic enzyme mixtures. Commercial enzymes (Accellerase 1000 +/- Multifect Xylanase, and Spezyme CP +/- Novozyme 188) were used to test the system and serve as comparative benchmarks. Using ammonia-fiber expansion (AFEX) pretreated corn stover ground to 0.5 mm and a glucan loading of 0.2%, an enzyme loading of 15 mg protein/g glucan, and 48 h digestion at 50 degrees C, commercial enzymes released 53% and 41% of the available glucose and xylose, respectively. Mixtures of three, five, and six pure enzymes of Trichoderma species, expressed in Pichia pastoris, were systematically optimized. Statistical models were developed for the optimization of glucose alone, xylose alone, and the average of glucose + xylose for two digestion durations, 24 and 48 h. The resulting models were statistically significant (P < 0.0001) and indicated an optimum composition for glucose release (values for optimized xylose release are in parentheses) of 29% (5%) cellobiohydrolase 1, 5% (14%) cellobiohydrolase 2, 25% (25%) endo-beta1,4-glucanase 1, 14% (5%) beta-glucosidase, 22% (34%) endo-beta1,4-xylanase 3, and 5% (17%) beta-xylosidase in 48 h at a protein loading of 15 mg/g glucan. Comparison of two AFEX-treated corn stover preparations ground to different particle sizes indicated that particle size (100 vs. 500 microm) makes a large difference in total digestibility. The assay platform and the optimized "core" set together provide a starting point for the rapid testing and optimization of alternate core enzymes from other microbial and recombinant sources as well as for the testing of "accessory" proteins for development of superior enzyme mixtures for biomass conversion.


Stereospecific structural perturbations arising from adenine N(6) butadiene triol adducts in duplex DNA.
Butadiene is oxidized in vivo to form stereoisomeric butadiene diol epoxides (BDE). These react with adenine N(6) in DNA yielding stereoisomeric N(6)-(2,3,4-trihydroxybutyl)-2'-deoxyadenosyl (BDT) adducts. When replicated in Escherichia coli, the (2R,3R)-N(6)-(2,3,4-trihydroxybutyl)-2'-deoxyadenosyl adduct yielded low levels of A-->G mutations whereas the (2S,3S)-N(6)-(2,3,4-trihydroxybutyl)-2'-deoxyadenosyl butadiene triol adduct yielded low levels of A-->C mutations [Carmical, J. R., Nechev, L. V., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2000) Environ. Mol. Mutagen. 35, 48-56]. Accordingly, the structure of the (2R,3R)-N(6)-(2,3,4-trihydroxybutyl)-2'-deoxyadenosyl adduct at position X(6) in d(CGGACXAGAAG).d(CTTCTTGTCCG), the ras61 R,R-BDT-(61,2) adduct, was compared to the corresponding structure for the (2S,3S)-N(6)-(2,3,4-trihydroxybutyl)-2'-deoxyadenosyl adduct in the same sequence, the ras61 S,S-BDT-(61,2) adduct. Both the R,R-BDT-(61,2) and S,S-BDT-(61,2) adducts are oriented in the major groove of the DNA, accompanied by modest structural perturbations. However, structural refinement of the two adducts using a simulated annealing restrained molecular dynamics (rMD) approach suggests stereospecific differences in hydrogen bonding between the hydroxyl groups located at the beta- and gamma-carbons of the BDT moiety, and T(17) O(4) of the modified base pair X(6).T(17). The rMD calculations predict hydrogen bond formation between the gamma-OH and the T(17) O(4) in the R,R-BDT-(61,2) adduct whereas in the S,S-BDT-(61,2) adduct, hydrogen bond formation is predicted between the beta-OH and the T(17) O(4). This difference positions the two adducts differently in the major groove. This may account for the differential mutagenicity of the two adducts and suggests that the two adducts may interact differentially with other DNA processing enzymes. With respect to mutagenesis in E. coli, the minimal perturbation of DNA induced by both major groove adducts correlates with their facile bypass by three E. coli DNA polymerases in vitro and may account for their weak mutagenicity [Carmical, J. R., Nechev, L. V., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2000) Environ. Mol. Mutagen. 35, 48-56].


Unplanned early hospital readmission among critical care survivors: a mixed methods study of patients and carers.
Many intensive care (ICU) survivors experience early unplanned hospital readmission, but the reasons and potential prevention strategies are poorly understood. We aimed to understand contributors to readmissions from the patient/carer perspective. This is a mixed methods study with qualitative data taking precedence. Fifty-eight ICU survivors and carers who experienced early unplanned rehospitalisation were interviewed. Thematic analysis was used to identify factors contributing to readmissions, and supplemented with questionnaire data measuring patient comorbidity and carer strain, and importance rating scales for factors that contribute to readmissions in other patient groups. Data were integrated iteratively to identify patterns, which were discussed in five focus groups with different patients/carers who also experienced readmissions. Major patterns and contexts in which unplanned early rehospitalisation occurred in ICU survivors were described. Interviews suggested 10 themes comprising patient-level and system-level issues. Integration with questionnaire data, pattern exploration and discussion at focus groups suggested two major readmission contexts. A 'complex health and psychosocial needs' context occurred in patients with multimorbidity and polypharmacy, who frequently also had significant psychological problems, mobility issues, problems with specialist aids/equipment and fragile social support. These patients typically described inadequate preparation for hospital discharge, poor communication between secondary/primary care, and inadequate support with psychological care, medications and goal setting. This complex multidimensional situation contrasted markedly with the alternative 'medically unavoidable' readmission context. In these patients medical issues/complications primarily resulted in hospital readmission, and the other issues were absent or not considered important. Although some readmissions are medically unavoidable, for many ICU survivors complex health and psychosocial issues contribute concurrently to early rehospitalisation. Care pathways that anticipate and institute anticipatory multifaceted support for these patients merit further development and evaluation.


The Impact of Racism on Child and Adolescent Health.
The American Academy of Pediatrics is committed to addressing the factors that affect child and adolescent health with a focus on issues that may leave some children more vulnerable than others. Racism is a social determinant of health that has a profound impact on the health status of children, adolescents, emerging adults, and their families. Although progress has been made toward racial equality and equity, the evidence to support the continued negative impact of racism on health and well-being through implicit and explicit biases, institutional structures, and interpersonal relationships is clear. The objective of this policy statement is to provide an evidence-based document focused on the role of racism in child and adolescent development and health outcomes. By acknowledging the role of racism in child and adolescent health, pediatricians and other pediatric health professionals will be able to proactively engage in strategies to optimize clinical care, workforce development, professional education, systems engagement, and research in a manner designed to reduce the health effects of structural, personally mediated, and internalized racism and improve the health and well-being of all children, adolescents, emerging adults, and their families.


Analysis of stop-gain and frameshift variants in human innate immunity genes.
Loss-of-function variants in innate immunity genes are associated with Mendelian disorders in the form of primary immunodeficiencies. Recent resequencing projects report that stop-gains and frameshifts are collectively prevalent in humans and could be responsible for some of the inter-individual variability in innate immune response. Current computational approaches evaluating loss-of-function in genes carrying these variants rely on gene-level characteristics such as evolutionary conservation and functional redundancy across the genome. However, innate immunity genes represent a particular case because they are more likely to be under positive selection and duplicated. To create a ranking of severity that would be applicable to innate immunity genes we evaluated 17,764 stop-gain and 13,915 frameshift variants from the NHLBI Exome Sequencing Project and 1,000 Genomes Project. Sequence-based features such as loss of functional domains, isoform-specific truncation and nonsense-mediated decay were found to correlate with variant allele frequency and validated with gene expression data. We integrated these features in a Bayesian classification scheme and benchmarked its use in predicting pathogenic variants against Online Mendelian Inheritance in Man (OMIM) disease stop-gains and frameshifts. The classification scheme was applied in the assessment of 335 stop-gains and 236 frameshifts affecting 227 interferon-stimulated genes. The sequence-based score ranks variants in innate immunity genes according to their potential to cause disease, and complements existing gene-based pathogenicity scores. Specifically, the sequence-based score improves measurement of functional gene impairment, discriminates across different variants in a given gene and appears particularly useful for analysis of less conserved genes.


Subunit and catalytic component stoichiometries of an in vitro reconstituted human pyruvate dehydrogenase complex.
The human pyruvate dehydrogenase complex (PDC) is a 9.5-megadalton catalytic machine that employs three catalytic components, i.e. pyruvate dehydrogenase (E1p), dihydrolipoyl transacetylase (E2p), and dihydrolipoamide dehydrogenase (E3), to carry out the oxidative decarboxylation of pyruvate. The human PDC is organized around a 60-meric dodecahedral core comprising the C-terminal domains of E2p and a noncatalytic component, E3-binding protein (E3BP), which specifically tethers E3 dimers to the PDC. A central issue concerning the PDC structure is the subunit stoichiometry of the E2p/E3BP core; recent studies have suggested that the core is composed of 48 copies of E2p and 12 copies of E3BP. Here, using an in vitro reconstituted PDC, we provide densitometry, isothermal titration calorimetry, and analytical ultracentrifugation evidence that there are 40 copies of E2p and 20 copies of E3BP in the E2p/E3BP core. Reconstitution with saturating concentrations of E1p and E3 demonstrated 40 copies of E1p heterotetramers and 20 copies of E3 dimers associated with the E2p/E3BP core. To corroborate the 40/20 model of this core, the stoichiometries of E3 and E1p binding to their respective binding domains were reexamined. In these binding studies, the stoichiometries were found to be 1:1, supporting the 40/20 model of the core. The overall maximal stoichiometry of this in vitro assembled PDC for E2p:E3BP:E1p:E3 is 40:20:40:20. These findings contrast a previous report that implicated that two E3-binding domains of E3BP bind simultaneously to a single E3 dimer (Smolle, M., Prior, A. E., Brown, A. E., Cooper, A., Byron, O., and Lindsay, J. G. (2006) J. Biol. Chem. 281, 19772-19780).


Relatedness influences signal reliability in evolving robots.
Communication is an indispensable component of animal societies, yet many open questions remain regarding the factors affecting the evolution and reliability of signalling systems. A potentially important factor is the level of genetic relatedness between signallers and receivers. To quantitatively explore the role of relatedness in the evolution of reliable signals, we conducted artificial evolution over 500 generations in a system of foraging robots that can emit and perceive light signals. By devising a quantitative measure of signal reliability, and comparing independently evolving populations differing in within-group relatedness, we show a strong positive correlation between relatedness and reliability. Unrelated robots produced unreliable signals, whereas highly related robots produced signals that reliably indicated the location of the food source and thereby increased performance. Comparisons across populations also revealed that the frequency for signal production-which is often used as a proxy of signal reliability in empirical studies on animal communication-is a poor predictor of signal reliability and, accordingly, is not consistently correlated with group performance. This has important implications for our understanding of signal evolution and the empirical tools that are used to investigate communication.


Visual search with negative slopes: the statistical power of numerosity guides attention.
Four experiments were performed to examine the hypothesis that abstract, nonspatial, statistical representations of object numerosity can be used for attentional guidance in a feature search task. Participants searched for an odd-colored target among distractors of one, two, or three other colors. An enduring advantage of large over small sets (i.e., negative slopes of search functions) was found, and this advantage grew with the number of colored subsets among distractors. The results of Experiments 1 and 2 showed that the negative slopes cannot be ascribed to the spatial grouping between distractors but can be partially explained by the spatial density of the visual sets. Hence, it appears that observers relied on numerosity of subsets to guide attention. Experiments 3a and 3b tested the processes within and between color subsets of distractors more precisely. It was found that the visual system collects numerosity statistics that can be used for guidance within each subset independently. However, each subset representation should be serially selected by attention. As attention shifts from one subset to another, the "statistical power" effects from every single subset are accumulated to provide a more pronounced negative slope.


An SNP-Based Linkage Map for Zebrafish Reveals Sex Determination Loci.
A surprising diversity of mechanisms controls sex determination of vertebrate organisms, even among closely related species. Both genetic and temperature-dependent systems of sex determination have been described in teleost fish. In the common zebrafish model organism, heteromorphic sex chromosomes are not observed, and the potential role of a genetic component of sex determination remains largely unknown. Here we report a genome-wide linkage study of sex determination in zebrafish using a novel SNP genetic map. We identified loci on zebrafish chromosomes 5 (LOD score 7.9) and 16 (LOD score 9.3) governing sex determination as a complex trait, rather than as an XY or ZW genetic system. Each of these loci contains a prominent candidate gene with a conserved role in sex determination across additional species that suggest potential mechanisms of sex determination in zebrafish. The chromosome 5 locus harbors dmrt1, a key gene in sex determination from fruit flies to humans; mutation of the human DMRT1 ortholog is a cause of complete sex reversal of XY individuals. The chromosome 16 locus harbors cyp21a2; mutation of the human CYP21A2 ortholog is one of the more common causes of pseudohermaphroditism. Mutation detection at each of these candidate genes within the zebrafish cross identified hypomorphic variants on the female-associated allele of each locus. The two loci together accounted for 16% of variance of the trait. Interacting environmental cues are likely to be an additional important component of sex determination in zebrafish.


Enhancement of Mechanical Properties and Testing of Nitinol Stents in Cerebral Aneurysm Simulation Models.
Stents are promising medical devices widely used in the prevention of cerebral aneurysm rupture. As the performance of stents depends on their mechanical properties and cell configuration, the aim of this study was to optimize the stent design and test the hemodynamic properties by using computational solid mechanics and computational fluid dynamics. In order to test their performance, computer-based cerebral aneurysm models that mimic the conditions present after implantation into the human brain were tested. The strut configuration selected was the closed-cell type, and nitinol was chosen as the material for stent manufacture because the innate characteristics of this material increase stent flexibility. Three ideal sample stent types with different cell configurations were manufactured. Computational solid mechanics analysis of the sample stents showed over 30% difference in flexibility between stents. Furthermore, using a cerebral aneurysm model simulation, we found that the stents eased the hemodynamic factors of the cerebral aneurysm and lessened the flow velocity influx into the sac. A decrease in flow velocity led to a 50-60% reduction in wall shear stress, which is expected to prevent aneurysm rupture under clinical conditions. Stent design optimization was carried out by simulation and electropolishing. Corrosion resistance and surface roughness were evaluated after electropolishing performed under variable conditions, but 40 V and 10 s were the most optimal.


Interindividual variability and co-regulation of DNA methylation differ among blood cell populations.
DNA methylation regulates gene expression in a cell-type specific way. Although peripheral blood mononuclear cells (PBMCs) comprise a heterogeneous cell population, most studies of DNA methylation in blood are performed on total mononuclear cells. In this study, we investigated high resolution methylation profiles of 58 CpG sites dispersed over eight immune response genes in multiple purified blood cells from healthy adults and newborns. Adjacent CpG sites showed methylation levels that were increasingly correlated in adult blood vs. cord blood. Thus, while interindividual variability increases from newborn to adult blood, the underlying methylation changes may not be merely stochastic, but seem to be orchestrated as clusters of adjacent CpG sites. Multiple linear regression analysis showed that interindividual methylation variability was influenced by distance of average methylation levels to the closest border (0 or 100%), presence of transcription factor binding sites, CpG conservation across species and age. Furthermore, CD4+ and CD14+ cell types were negative predictors of methylation variability. Concerns that PBMC methylation differences may be confounded by variations in blood cell composition were justified for CpG sites with large methylation differences across cell types, such as in the IFN-γ gene promoter. Taken together, our data suggest that unsorted mononuclear cells are reasonable surrogates of CD8+ and, to a lesser extent, CD4+ T cell methylation in adult peripheral, but not in neonatal, cord blood.


Red blood cell Li+-Na+ countertransport, Na+-K+ cotransport, and the hemodynamics of hypertension.
Red blood cell Li+-Na+ countertransport and Na+-K+ cotransport activities, home blood pressure, invasive systemic hemodynamics, and limb venous compliance were measured in 65 white men (23 normotensive, 22 borderline hypertensive, and 20 mild essential hypertensive subjects). Li+-Na+ countertransport activity was positively and significantly correlated with subject-determined home systolic blood pressure (r = 0.31, p less than 0.02) and with directly measured systolic (r = 0.29, p less than 0.02) and diastolic (r = 0.27, p less than 0.03) blood pressures in the hemodynamic laboratory, independent of potential confounding variables. Analysis of the hemodynamic determinants of blood pressure revealed a significant positive correlation of countertransport with vascular resistance (r = 0.30, p less than 0.02) but not with cardiac output or cardiac index. High red blood cell Na+-K+ cotransport activity was not independently associated with hypertension or with a characteristic hemodynamic pattern but was related to decreased venous compliance. Red blood cell Li+-Na+ countertransport deserves further study as a marker for the genetic substrate of human essential hypertension. Red cell Na+-K+ cotransport may be altered secondarily by factors related to high blood pressure and seems to be a valid marker for abnormalities of the venous system in hypertension.


Trehalose biosynthesis is involved in sporulation of Stagonospora nodorum.
Stagonospora nodorum is a necrotrophic fungal pathogen that is the causal agent of leaf and glume blotch on wheat. S. nodorum is a polycyclic pathogen, whereby rain-splashed pycnidiospores attach to and colonise wheat tissue and subsequently sporulate again within 2-3weeks. As several cycles of infection are needed for a damaging infection, asexual sporulation is a critical phase of its infection cycle. A non-targeted metabolomics screen for sporulation-associated metabolites identified that trehalose accumulated significantly in concert with asexual sporulation both in vitro and in planta. A reverse-genetics approach was used to investigate the role of trehalose in asexual sporulation. Trehalose biosynthesis was disrupted by deletion of the gene Tps1, encoding a trehalose 6-phosphate synthase, resulting in almost total loss of trehalose during in vitro growth and in planta. In addition, lesion development and pycnidia formation were also significantly reduced in tps1 mutants. Reintroduction of the Tps1 gene restored trehalose biosynthesis, pathogenicity and sporulation to wild-type levels. Microscopic examination of tps1 infected wheat leaves showed that pycnidial formation often halted at an early stage of development. Further examination of the tps1 phenotype revealed that tps1 pycnidiospores exhibited a reduced germination rate while under heat stress, and tps1 mutants had a reduced growth rate while under oxidative stress. This study confirms a link between trehalose biosynthesis and pathogen fitness in S.nodorum.


SAS macros for testing statistical mediation in data with binary mediators or outcomes.
Statistical mediation is an important tool in behavioral health sciences, but it has been confined primarily to continuous variables. As prevention studies become increasingly common, more often the mediator or outcome is binary. Recent work by D. P. MacKinnon and J. H. Dwyer (1993) has explicated the steps necessary to estimate models for mediation when the mediator or the outcome is binary. To report the release of a set of SAS macros used to implement the statistical analyses required to analyze data with binary and continuous-level data. A brief introduction to the methodology of mediation analysis in the presence of a binary outcome, mediator, or both is provided. The macros are tested on a sample of 84 participants who were experiencing pain. It is hypothesized that the relationship between pain and fatigue is mediated by sleep disturbance. The relationship between pain and fatigue was mediated by the presence of sleep disturbances, and the amount of mediation was 23.34%. The SAS macros are available for download without charge from the second author's Web site. Instructions are provided in an included technical manual.


Dithiol amino acids can structurally shape and enhance the ligand-binding properties of polypeptides.
The disulfide bonds that form between two cysteine residues are important in defining and rigidifying the structures of proteins and peptides. In polypeptides containing multiple cysteine residues, disulfide isomerization can lead to multiple products with different biological activities. Here, we describe the development of a dithiol amino acid (Dtaa) that can form two disulfide bridges at a single amino acid site. Application of Dtaas to a serine protease inhibitor and a nicotinic acetylcholine receptor inhibitor that contain disulfide constraints enhanced their inhibitory activities 40- and 7.6-fold, respectively. X-ray crystallographic and NMR structure analysis show that the peptide ligands containing Dtaas have retained their native tertiary structures. We furthermore show that replacement of two cysteines by Dtaas can avoid the formation of disulfide bond isomers. With these properties, Dtaas are likely to have broad application in the rational design or directed evolution of peptides and proteins with high activity and stability.


Division of labor in honey bee gut microbiota for plant polysaccharide digestion.
Bees acquire carbohydrates from nectar and lipids; and amino acids from pollen, which also contains polysaccharides including cellulose, hemicellulose, and pectin. These potential energy sources could be degraded and fermented through microbial enzymatic activity, resulting in short chain fatty acids available to hosts. However, the contributions of individual microbiota members to polysaccharide digestion have remained unclear. Through analysis of bacterial isolate genomes and a metagenome of the honey bee gut microbiota, we identify that Bifidobacterium and Gilliamella are the principal degraders of hemicellulose and pectin. Both Bifidobacterium and Gilliamella show extensive strain-level diversity in gene repertoires linked to polysaccharide digestion. Strains from honey bees possess more such genes than strains from bumble bees. In Bifidobacterium, genes encoding carbohydrate-active enzymes are colocated within loci devoted to polysaccharide utilization, as in Bacteroides from the human gut. Carbohydrate-active enzyme-encoding gene expressions are up-regulated in response to particular hemicelluloses both in vitro and in vivo. Metabolomic analyses document that bees experimentally colonized by different strains generate distinctive gut metabolomic profiles, with enrichment for specific monosaccharides, corresponding to predictions from genomic data. The other 3 core gut species clusters (Snodgrassella and 2 Lactobacillus clusters) possess few or no genes for polysaccharide digestion. Together, these findings indicate that strain composition within individual hosts determines the metabolic capabilities and potentially affects host nutrition. Furthermore, the niche specialization revealed by our study may promote overall community stability in the gut microbiomes of bees.


Non-genic transcription at the Drosophila bithorax complex functional activity of the dark matter of the genome.
Drosophila melanogaster is a powerful model system for the study of gene regulation due to its short generation time, high fertility and the availability of various genetic tools to manipulate the genome. Investigation into the regulation of homeotic genes and their role in embryonic patterning during development was pioneered in Drosophila. Recently, the molecular mechanisms responsible for regulating gene expression in the bithorax complex have been the focus of active study. Many of these studies have pointed to the importance of cis-regulatory modules, genetic sequences that direct the temporal and spatial patterns of gene expression over large genomic distances. Additional components of the regulatory code have emerged beyond the primary DNA sequence. In particular, non-genic transcription is an important mechanism for controlling gene expression either through direct transcriptional mechanisms that mediate dynamic epigenetic control of the chromatin environment or through functional activity of the RNA products.


Vehicle characteristics associated with LATCH use and correct use in real-world child restraint installations.
The objective of this study was to determine if vehicle features associated with LATCH ease-of-use in laboratory studies with volunteers predict LATCH use and misuse in real-world child restraint installations. Vehicle characteristics were extracted from prior surveys of more than 100 top-selling 2010-13 vehicles. Use and correct use of LATCH was determined from records of more than 14,000 child restraint installations in these vehicles that were inspected by child passenger safety technicians at Safe Kids car seat checkup events during 2010-12. Logistic regression was used to examine the association between vehicle features and use and correct use of lower anchors and top tethers, controlling for other relevant installation features. Lower anchors were more likely to be used and correctly used when the clearance angle around them was greater than 54°, the force required to attach them to the lower anchors was less than 178N, and their depth within the seat bight was less than 4cm. Restraints were more likely to be attached correctly when installed with the lower anchors than with the seat belt. After controlling for lower anchor use and other installation features, the likelihood of tether use and correct use in installations of forward-facing restraints was significantly higher when there was no hardware present that could potentially be confused with the tether anchor or when the tether anchor was located on the rear deck, which is typical in sedans. There is converging evidence from laboratory studies with volunteers and real-world child restraint installations that vehicle features are associated with correct LATCH use. Vehicle designs that improve the ease of installing child restraints with LATCH could improve LATCH use rates and reduce child restraint misuse.


Role of sodium-calcium exchanger in modulating the action potential of ventricular myocytes from normal and failing hearts.
Increased Na+-Ca2+ exchange (NCX) activity in heart failure and hypertrophy may compensate for depressed sarcoplasmic reticular Ca2+ uptake, provide inotropic support through reverse-mode Ca2+ entry, and/or deplete intracellular Ca2+ stores. NCX is electrogenic and depends on Na+ and Ca2+ transmembrane gradients, making it difficult to predict its effect on the action potential (AP). Here, we examine the effect of [Na+]i on the AP in myocytes from normal and pacing-induced failing canine hearts and estimate the direction of the NCX driving force using simultaneously recorded APs and Ca2+ transients. AP duration shortened with increasing [Na+]i and was correlated with a shift in the reversal point of the NCX driving force. At [Na+]i > or =10 mmol/L, outward NCX current during the plateau facilitated repolarization, whereas at 5 mmol/L [Na+]i, NCX had a depolarizing effect, confirmed by partially inhibiting NCX with exchange inhibitory peptide. Exchange inhibitory peptide shortened the AP duration at 5 mmol/L [Na+]i and prolonged it at [Na+]i > or =10 mmol/L. With K+ currents blocked, total membrane current was outward during the late plateau of an AP clamp at 10 mmol/L [Na+]i and became inward close to the predicted reversal point for the NCX driving force. The results were reproduced using a computer model. These results indicate that NCX plays an important role in shaping the AP of the canine myocyte, helping it to repolarize at high [Na+]i, especially in the failing heart, but contributing a depolarizing, potentially arrhythmogenic, influence at low [Na+]i.


[Brazilian guideline for the treatment of patients with opioids dependence syndrome].
There is a relatively low prevalence of opioid use in Brazil, particularly involving the non-medical use of codeine and opiate-containing syrups. However, opioid dependence syndrome shows a significant total impact on mortality and morbidity. Over the past 20 years, scientific progress has changed our understanding of the nature of opioid addiction and its various possible treatments. Addiction is a chronic illness treatable if the treatment is well-delivered and tailored to the needs of the particular patient. There is indeed an array of treatments that can effectively reduce drug use, help manage drug cravings, prevent relapses and restore people to productive social functioning. The treatment of drug addiction will be part of long-term, medical, psychological, and social perspectives. This guideline aims at providing guidance to psychiatrists and other mental health professionals who care for patients with opioid dependence syndrome. It comments on the somatic and psychosocial treatment that is used for such patients, and reviews scientific evidences and their strength. Also, the essential historical, epidemiological and neurobiological aspects of opioid dependence are reviewed.


Development and qualification of the parallel line model for the estimation of human influenza haemagglutinin content using the single radial immunodiffusion assay.
Infection with human influenza virus leads to serious respiratory disease. Vaccination is the most common and effective prophylactic measure to prevent influenza. Influenza vaccine manufacturing and release is controlled by the correct determination of the potency-defining haemagglutinin (HA) content. This determination is historically done by single radial immunodiffusion (SRID), which utilizes a statistical slope-ratio model to estimate the actual HA content. In this paper we describe the development and qualification of a parallel line model for analysis of HA quantification by SRID in cell culture-derived whole virus final monovalent and trivalent influenza vaccines. We evaluated plate layout, sample randomization, and validity of data and statistical model. The parallel line model was shown to be robust and reproducible. The precision studies for HA content demonstrated 3.8-5.0% repeatability and 3.8%-7.9% intermediate precision. Furthermore, system suitability criteria were developed to guarantee long-term stability of this assay in a regulated production environment. SRID is fraught with methodological and logistical difficulties and the determination of the HA content requires the acceptance of new and modern release assays, but until that moment, the described parallel line model represents a significant and robust update for the current global influenza vaccine release assay.


Mortality and cancer incidence in Korean patients with systemic lupus erythematosus: results from the Hanyang lupus cohort in Seoul, Korea.
The rates of mortality and cancer have not been compared between Korean systemic lupus erythematosus (SLE) patients and the general population. The objective of this study was to compare the observed mortality and cancer incidence rates of SLE patients with the expected age-, sex- and calendar-matched incidence rates of a comparable general control population. This study included 466 SLE patients who were in the Hanyang Lupus Cohort, Seoul and we excluded the males from the analyses because the number of males was too small to expect any incidence of cancer or death. We used the national female mortality rates from 1992 to 2001 as recorded by the Korean National Statistical Office as the standard mortality rates, and the Seoul Cancer Registry data as the standard cancer incidence rate. Ten of the females in our study died, giving a crude mortality rate of 596.0/100 000 person-years (PY) and a standardized mortality ratio (SMR) of 3.02. The crude cancer incidence was 179.3/100 000 PYand the standard incidence ratio was 1.04. These results demonstrate that the SMR is significantly higher in SLE patients than in the general population, whereas the incidence of cancer is the same in these two groups.


High GATA2 expression is a poor prognostic marker in pediatric acute myeloid leukemia.
In acute myeloid leukemia (AML), aberrant expression and mutations of transcription factors have been correlated with disease outcome. In the present study, we performed expression and mutation screening of GATA2, which is an essential transcription factor for regulation of myeloid lineage determination, in de novo pediatric AML patients. GATA2 mutations were detected in 5 of 230 patients, representing a frequency of 2.2% overall and 9.8% in cytogenetically normal AML. GATA2 expression analysis demonstrated that in 155 of 237 diagnostic samples (65%), GATA2 expression was higher than in normal BM. In complete remission, normalization of GATA2 expression was observed, whereas GATA2 expression levels stayed high in patients with resistant disease. High GATA2 expression at diagnosis was an independent poor prognostic factor for overall survival (hazard ratio [HR] = 1.7, P = .045), event-free survival (HR = 2.1, P = .002), and disease-free survival (HR = 2.3, P = .004). The prognostic impact of GATA2 was particularly evident in specific AML subgroups. In patients with French-American-British M5 morphology, inv(16), or high WT1 expression, significant differences in survival were observed between patients with high versus normal GATA2 expression. We conclude that high GATA2 expression is a novel poor prognostic marker in pediatric AML, which may contribute to better risk-group stratification and risk-adapted therapy in the future.


Macular impairment in mitochondrial diseases: a potential biomarker of disease severity.
The high-energy demands of the retina are thought to contribute to its particular vulnerability to mitochondrial dysfunction. Photoreceptors are the cells with the higher oxygen consumption within the retina, and among these, the cones contain more mitochondria and have a higher energy demand than rods. A cohort of twenty-two patients with genetically-defined mitochondrial diseases (MDs) were enrolled to determine if the macula is functionally and anatomically impaired in these metabolic disorders. Visual acuity and fERG amplitude of patients with primary mitochondrial dysfunction were reduced compared to controls. Furthermore, SD-OCT layer segmentation showed a reduction of retinal and outer nuclear layer (ONL) volume in the macula of the patients. fERG amplitude showed a positive correlation with both ONL volume and thickness. A negative relationship was noted between fERG amplitude and disease severity assessed with Newcastle Mitochondrial Disease Adult Scale. In conclusion, MDs are associated with functional and anatomical alteration of macular cone system, characterized by its strong correlation with clinical disease severity suggesting a role as a potential biomarker of primary mitochondrial disorders.


Differential activation of pp60(c-src) and pp62(c-yes) in human colorectal carcinoma liver metastases.
pp60(c-src) and pp62(c-yes) are protein tyrosine kinases whose specific activities are increased in primary colorectal carcinomas. Activity of pp60(c-src) is further increased in colorectal liver metastases. This study was undertaken to compare pp60(c-src) and pp62(c-yes) expression and activity in human colorectal carcinoma liver metastases and to determine the potential prognostic significance of differences in activation of these two kinases. The pp60(c-src) and pp62(c-yes) tyrosine kinase activities and protein levels relative to those in normal colonic mucosa were determined using an immune complex kinase assay and immunoblot analysis in tissue specimens from 22 patients with primary colorectal carcinoma and synchronous metastatic liver disease and from 9 patients with metachronous colorectal carcinoma liver metastases. Of the primary colon tumors, 64% of the tumors contained elevated activities of both pp60(c-src) and pp62(c-yes). For liver metastases, however, only 10% had activation of both tyrosine kinases, 61% had elevated pp60(c-src) activity only, and 23% had elevated pp62(c-yes) activity only. Analysis of synchronous metastases from primary tumors with elevated activities in both kinases demonstrated that in 71% of these patients, the activity of either pp60(c-src) or pp62(c-yes) decreases relative to the primary tumor. Protein levels of pp60(c-src) and pp62(c-yes) in primary carcinomas and metastases remained unchanged from levels in normal colonic mucosa. These results demonstrate that differential regulation of the activities of pp60(c-src) and pp62(c-yes) occurs during tumor progression. Patients with either synchronous or metachronous liver metastases and elevated pp62(c-yes) kinase activity have biologically more aggressive disease and a worse prognosis than patients without elevated pp62(c-yes) activity in their liver metastases (median survival, 13 months versus 30 months, P < 0.005, Wilcoxon signed rank test). Analysis of patients with synchronous liver metastases also demonstrated a worse prognosis for those with elevated pp62(c-yes) kinase activity (P < 0.05, Wilcoxon signed rank test).


Roles of CD9 molecules in survival and activation of human eosinophils.
CD9 is a cell surface glycoprotein belonging to the transmembrane 4 superfamily. In this report, we demonstrate that cross-linking CD9 with different forms of mAb activates distinct functions of human eosinophils. Anti-CD9 mAb (clone ALB6) immobilized onto tissue culture plates induced eosinophil degranulation. This effect of anti-CD9 mAb (clone ALB6) was unique because other immobilized clones of anti-CD9 mAb (clones FMC56 and ML13) or anti-HLA Class I mAb failed to induce degranulation. In addition, neutrophil degranulation was not provoked by the immobilized clone ALB6, consistent with a lack of expression of CD9 on neutrophils. Eosinophil degranulation induced by anti-CD9 mAb (clone ALB6) was abolished by pretreatment of eosinophils with anti-CD18 mAb, suggesting that beta2 integrins are involved in the reaction. In contrast to the results with immobilized clones, all clones of anti-CD9 mAb in soluble form enhanced eosinophil survival. This enhanced survival was inhibited by anti-granulocyte-macrophage-CSF (GM-CSF) mAb, suggesting that autocrine production of GM-CSF by eosinophils mediated the enhanced survival. In fact, by ELISA and RT-PCR, GM-CSF protein and mRNA were detected in supernatants and cell lysates of eosinophils stimulated by soluble anti-CD9 mAb, but not by immobilized mAb. These results indicate that CD9 serves as a molecule that delivers stimulation signals on eosinophils. The eosinophil's cellular responses may differ and be dependent on the manner of receptor ligation.


E2 multimeric scaffold for vaccine formulation: immune response by intranasal delivery and transcriptome profile of E2-pulsed dendritic cells.
The E2 multimeric scaffold represents a powerful delivery system able to elicit robust humoral and cellular immune responses upon systemic administrations. Here recombinant E2 scaffold displaying the third variable loop of HIV-1 Envelope gp120 glycoprotein was administered via mucosa, and the mucosal and systemic immune responses were analysed. To gain further insights into the molecular mechanisms that orchestrate the immune response upon E2 vaccination, we analysed the transcriptome profile of dendritic cells (DCs) exposed to the E2 scaffold with the aim to define a specific gene expression signature for E2-primed immune responses. The in vivo immunogenicity and the potential of E2 scaffold as a mucosal vaccine candidate were investigated in BALB/c mice vaccinated via the intranasal route. Fecal and systemic antigen-specific IgA antibodies, cytokine-producing CD4(+) and CD8(+) cells were induced assessing the immunogenicity of E2 particles via intranasal administration. The cytokine analysis identified a mixed T-helper cell response, while the systemic antibody response showed a prevalence of IgG1 isotype indicative of a polarized Th2-type immune response. RNA-Sequencing analysis revealed that E2 scaffold up-regulates in DCs transcriptional regulators of the Th2-polarizing cell response, defining a type 2 DC transcriptomic signature. The current study provides experimental evidence to the possible application of E2 scaffold as antigen delivery system for mucosal immunization and taking advantages of genome-wide approach dissects the type of response induced by E2 particles.


Evolution of plasmid-coded resistance to broad-spectrum cephalosporins.
A clinical isolate of Klebsiella ozaenae with transferable resistance to broad-spectrum cephalosporins produces a beta-lactamase determined by plasmid pBP60. The beta-lactamase had the same isoelectric point as SHV-1 (7.6). From heteroduplex analysis, an extensive homology between the two bla genes could be deduced; therefore, the new beta-lactamase was designated SHV-2. Enzymatic studies revealed that SHV-2 was able to hydrolyze broad-spectrum cephalosporins due to an increased affinity of these compounds for the enzyme. The assumption that SHV-2 is a natural mutant of SHV-1 was strongly supported by the isolation of a laboratory mutant of SHV-1 that showed activities similar to those of SHV-2.


Brief psychotherapy in the treatment of anorexia nervosa. Outcome at one year.
Thirty out-patients with severe anorexia nervosa were randomly allocated to either 12 sessions of dietary advice or 12 sessions of combined individual and family psychotherapy. At one-year follow-up both groups showed significant overall improvement, and the dietary advice group showed significant weight gain. A similar mean weight gain for the psychotherapy patients did not reach statistical significance, but this group made significant improvements in sexual and social adjustment.


Low European methyl chloroform emissions inferred from long-term atmospheric measurements.
Methyl chloroform (CH3CCl3, 1,1,1,-trichloroethane) was used widely as a solvent before it was recognized to be an ozone-depleting substance and its phase-out was introduced under the Montreal Protocol. Subsequently, its atmospheric concentration has declined steadily and recent European methyl chloroform consumption and emissions were estimated to be less than 0.1 gigagrams per year. However, data from a short-term tropospheric measurement campaign (EXPORT) indicated that European methyl chloroform emissions could have been over 20 gigagrams in 2000 (ref. 6), almost doubling previously estimated global emissions. Such enhanced emissions would significantly affect results from the CH3CC13 method of deriving global abundances of hydroxyl radicals (OH) (refs 7-12)-the dominant reactive atmospheric chemical for removing trace gases related to air pollution, ozone depletion and the greenhouse effect. Here we use long-term, high-frequency data from Mace Head, Ireland and Jungfraujoch, Switzerland, to infer European methyl chloroform emissions. We find that European emission estimates declined from about 60 gigagrams per year in the mid-1990s to 0.3-1.4 and 1.9-3.4 gigagrams per year in 2000-03, based on Mace Head and Jungfraujoch data, respectively. Our European methyl chloroform emission estimates are therefore higher than calculated from consumption data, but are considerably lower than those derived from the EXPORT campaign in 2000 (ref. 6).


Prolonged Versus Short Infusion Rates of IV Magnesium in Hospitalized General Medicine Patients with Hypomagnesemia.
Introduction: Due to the renal handling mechanism of magnesium, prolonging the time for infusion of intravenous (IV) magnesium has been postulated to decrease magnesium requirements; however, a paucity of clinical evidence exists to support prolonging infusion rates. Objective: To assess if there is a difference in magnesium replacement required in the medicine population at an academic medical center when prolonged infusion rates (0.5 g/h) are compared to short infusion rates of > 0.5 g/h. Methods: A retrospective chart review was performed before and after implementation of the hypomagnesemia protocol (November 2015). Patients who received at least one dose of IV magnesium during hospitalization were selected from general medicine units. Primary aim was to determine if a difference exists in percent of days IV magnesium repletion required between patients receiving prolonged versus short infusion rates. Secondary objectives were to determine if a difference exists in total grams of magnesium received, percent of days magnesium levels were maintained in the optimal (1.4-2.7) and desired (2-2.7) therapeutic ranges, and incidence of hypomagnesemia (< 1.4 g/dL) and hypermagnesemia (> 2.7 g/dL). For safety, incidence of hypotension (systolic blood pressure < 90/60 mm Hg) during the magnesium infusion was recorded. Results: Totally, 45 patients were included in each cohort for a total of 90 patients to meet power. No differences existed between protocol groups for any demographic variables (all P > .05). Median infusion rate for the short infusion cohort was 1.8 g/h (range 1-2 g/h). Percent of days IV magnesium was replaced was 34.8% versus 37.8% (P = .39) in the short and prolonged infusion groups, respectively. No difference existed between groups for secondary outcomes (all P > .05). Conclusion: Prolonged magnesium infusion rates did not decrease magnesium replacement requirements. These results have been used to propose revision of our current magnesium infusion protocol to reduce infusion length.


The bacteriology of biopsies differs between newly diagnosed, untreated, Crohn's disease and ulcerative colitis patients.
The bacterial community (microbiota) that inhabits the gut of humans appears to be an important source of antigens that drive the chronic immunological processes characteristic of Crohn's disease (CD) and ulcerative colitis (UC). Most of the members of the microbiota have not yet been cultured, but nucleic-acid-based methods of detection and enumeration can provide information about the community. This investigation used these methods to obtain information about the bacteria associated with mucosal surfaces in the gut of 20 CD and 15 UC patients. Biopsies were collected from inflamed and non-inflamed sites in the intestines of newly diagnosed, untreated patients. Biopsies were also collected from several intestinal sites of 14 healthy subjects. The bacterial collections associated with the biopsies were analysed by generating PCR/denaturing gradient gel electrophoresis (DGGE) profiles, the preparation of 16S rRNA gene clone libraries, and qualitative PCR to detect specific groups of bacteria. The total numbers of bacteria associated with the biopsies were determined by real-time quantitative PCR. DGGE profiles generated from the terminal ileum and various colonic regions were characteristic of each individual but differed between subjects. DGGE profiles and 16S rRNA gene libraries showed that the bacteria associated with inflamed and non-inflamed tissues did not differ. UC patients had more bacteria associated with biopsies than did CD patients (P<0.01). Statistical analysis of the composition of 16S rRNA gene libraries showed that the bacterial collections in UC and CD patients differed (P<0.05). Unclassified members of the phylum Bacteroidetes were more prevalent in CD than in UC patients. Therefore, the types and numbers of bacteria associated with biopsy samples were distinctly different for UC and CD patients. The observations made in this study should permit targeting of specific bacteriological abnormalities in investigations of the pathogenesis of inflammatory bowel diseases and provide targets for medical interventions.


The protective effect of glycyrrhetinic acid on carbon tetrachloride-induced chronic liver fibrosis in mice via upregulation of Nrf2.
This study was designed to investigate the potentially protective effects of glycyrrhetinic acid (GA) and the role of transcription factor nuclear factor-erythroid 2(NF-E2)-related factor 2 (Nrf2) signaling in the regulation of Carbon Tetrachloride (CCl(4))-induced chronic liver fibrosis in mice. The potentially protective effects of GA on CCl(4)-induced chronic liver fibrosis in mice were depicted histologically and biochemically. Firstly, histopathological changes including regenerative nodules, inflammatory cell infiltration and fibrosis were induced by CCl(4).Then, CCl(4) administration caused a marked increase in the levels of serum aminotransferases (GOT, GPT), serum monoamine oxidase (MAO) and lipid peroxidation (MDA) as well as MAO in the mice liver homogenates. Also, decreased nuclear Nrf2 expression, mRNA levels of its target genes such as superoxide dismutase 3 (SOD3), catalase (CAT), glutathione peroxidase 2 (GPX2), and activity of cellular antioxidant enzymes were found after CCl(4) exposure. All of these phenotypes were markedly reversed by the treatment of the mice with GA. In addition, GA exhibited the antioxidant effects in vitro by on FeCl(2)-ascorbate induced lipid peroxidation in mouse liver homogenates, and on DPPH scavenging activity. Taken together, these results suggested that GA can protect the liver from oxidative stress in mice, presumably through activating the nuclear translocation of Nrf2, enhancing the expression of its target genes and increasing the activity of the antioxidant enzymes. Therefore, GA may be an effective hepatoprotective agent and viable candidate for treating liver fibrosis and other oxidative stress-related diseases.


Therapy in virtual environments--clinical and ethical issues.
As virtual reality and computer-assisted therapy strategies are increasingly implemented for the treatment of psychological disorders, ethical standards and guidelines must be considered. This study determined a set of ethical and legal guidelines for treatment of post-traumatic stress disorder (PTSD)/traumatic brain injury (TBI) in a virtual environment incorporating the rights of an individual who is represented by an avatar. A comprehensive literature review was undertaken. An example of a case study of therapy in Second Life (a popular online virtual world developed by Linden Labs) was described. Ethical and legal considerations regarding psychiatric treatment of PTSD/TBI in a virtual environment were examined. The following issues were described and discussed: authentication of providers and patients, informed consent, patient confidentiality, patient well-being, clinician competence (licensing and credentialing), training of providers, insurance for providers, the therapeutic environment, and emergencies. Ethical and legal guidelines relevant to these issues in a virtual environment were proposed. Ethical and legal issues in virtual environments are similar to those that occur in the in-person world. Individuals represented by an avatar have the rights equivalent to the individual and should be treated as such.


Signal peptide recognition in Trypanosoma cruzi GP82 adhesin relies on its localization at protein N-terminus.
Trypanosoma cruzi, the causative agent of Chagas disease, has a dense coat of GPI-anchored virulence factors. T. cruzi GPI-anchored adhesin GP82 is encoded by a repertoire of transcripts containing several in-frame initiation codons located up-stream from that adjacent to the predicted signal peptide (SP). Transfection of T. cruzi epimastigotes with constructs encoding GP82 starting at the SP or from the farthest up-stream methionine confirmed protein expression on the parasite cell surface, comparable to the native GP82. Proteins were fully functional, inducing parasite adhesion to HeLa cells and lysosome mobilization, events required for parasite invasion. Transgenic and native GP82 proteins showed indistinguishable electrophoretic mobility, suggesting similar processing of the SP. Deletion of SP generated a ~72 kDa protein devoid of N-linked oligosaccharides allowing irrefutable identification of GP82 precursor. SP transposition to an internal region of GP82 rendered the signal unrecognizable by the signal peptidase and incapable to direct the nascent protein for ER-membrane association. Altogether our data strongly suggests that GP82 SP fails to function as transmembrane domain and its recognition by the signal peptidase shows strict dependence on the signal localization at protein N-terminus. This report presents the first experimental characterization of the full-length GP82 and its signal peptide.


MiR-99b-5p expression and response to tyrosine kinase inhibitor treatment in clear cell renal cell carcinoma patients.
A number of treatments targeting VEGF or mTOR pathways have been approved for metastatic clear cell Renal Cell Carcinoma (ccRCC), but the majority of patients show disease progression after first line therapy with a very low rate of complete or long-term responders. It has been shown that miRs may play a role in prediction of treatment response in various cancer types. The aim of our study was to identify a miR signature predictive for RCC patients' response to antiangiogenic tyrosine kinase inhibitor (TKI) treatment in the first line therapy. Sequencing of 40 paired normal/tumor formalin fixed and paraffin embedded ccRCC tissues revealed separate clustering via unsupervised dendrograms. With supervised analysis, the strongest differential expression was obtained with miR-99b-5p, which was significantly lower in patients with short progression free survival (<8 months) and TKI non-responders (progressive disease patients according to RECIST) (p<0.0001, each). Validation using RTqPCR and a second patient cohort compiled from three different hospitals (n=65) showed higher expression of miR-99b-5p in complete responders, but this trend did not reach statistical significance. It is concluded that low miR-99b-5p expression analyzed with sequencing methodology may correlate with tumor progression in TKI-treated ccRCC patients.


Antidepressant agents for the treatment of chronic pain and depression.
Depression and painful somatic symptoms commonly occur together. Depression and chronic pain can have devastating effects on a patient's health, productivity, and overall quality of life. When moderate-to-severe pain exists, it can impair patient function while making treatment more difficult or resistant, with increased severity in depressive symptoms and worse outcomes. A variety of chronic pain syndromes exist, including diabetic neuropathy. A high prevalence of patients with chronic pain display depressive symptoms. Treatment for these conditions relies on pharmacologic therapy coupled with diligent, periodic assessments of changes in symptom severity. The link between pain and depression lies in the central and peripheral nervous systems. The brain stem serves as an important connection between the higher brain centers and the spinal cord. In the brain stem, the neurotransmitters serotonin and norepinephrine modulate pain transmission through ascending and descending neural pathways. Both serotonin and norepinephrine are also key neurotransmitters involved with the pathophysiology of depression. Tricyclic antidepressants are effective treatments for pain and depression; selective serotonin reuptake inhibitors provide less benefit. Duloxetine and venlafaxine, which are serotonin and norepinephrine reuptake inhibitors, were shown in clinical trials to alleviate pain and depressive symptoms. Diabetic neuropathy and other chronic pain syndromes were also shown to benefit from duloxetine and venlafaxine. Antidepressants remain fundamental therapeutic agents for depression and anxiety disorders. Their extended use into chronic pain, depression with physical pain, physical pain with or without depression, and other potential medical conditions should be recognized.


Use of real-time polymerase chain reaction to measure gene expression in single cells.
Quantification of the expression of individual genes can reveal much concerning the processes occurring within a cell. In the vast majority of cases, activation or repression of a gene is indicative of altered utilization of the pathway or process in which it functions. Although microarray analysis has the power to provide data concerning the expression of thousands of genes in a single experiment, validation of results using alternative methods is still essential. This is particularly true if the amount of RNA available for microarray analysis is very small, necessitating methods of RNA amplification. The gold-standard for quantifying mRNA transcripts from an individual gene is the use of reverse transcription followed by real-time PCR. This approach has yielded highly accurate and reproducible data, even when applied to minute samples, such as single oocytes or single embryos. This chapter describes protocols for the quanitification of mRNA transcripts using real-time PCR and considers issues specific to analysis of single cells.


Investing in health: is social housing value for money? A cost-utility analysis.
There is a healthy public policy agenda investigating the health impacts of improving living conditions. However, there are few economic evaluations, to date, assessing value for money. We conducted the first cost-effectiveness analysis of a nationwide intervention transferring social and private tenants to new-build social housing, in Scotland. A quasi-experimental prospective study was undertaken involving 205 intervention households and 246 comparison households, over 2 years. A cost-utility analysis assessed the average cost per change in health utility (a single score summarising overall health-related quality of life), generated via the SF-6D algorithm. Construction costs for new builds were included. Analysis was conducted for all households, and by family, adult and elderly households; with estimates adjusted for baseline confounders. Outcomes were annuitised and discounted at 3.5%. The average discounted cost was £18, 708 per household, at a national programme cost of £ 28.4 million. The average change in health utility scores in the intervention group attributable to the intervention were +0.001 for all households, +0.001 for family households, -0.04 for adult households and -0.03 for elderly households. All estimates were statistically insignificant. At face value, the interventions were not value for money in health terms. However, because the policy rationale was the amenity provision of housing for disadvantaged groups, impacts extend beyond health and may be fully realised over the long term. Before making general value-for-money inferences, economic evaluation should attempt to estimate the full social value of interventions, model long-term impacts and explicitly incorporate equity considerations.


Evaluation of the Genotype® MTBDRplus assay as a tool for drug resistance surveys.
A national tuberculosis (TB) drug resistance survey in Tanzania. To compare the performance of the Genotype® MTBDRplus line-probe assay (LPA) on smear-positive sputum specimens with conventional culture and isoniazid (INH) plus rifampicin (RMP) drug susceptibility testing (DST). Mycobacterium tuberculosis isolates tested at the Tanzanian Central TB Reference Laboratory (CTRL) were submitted for quality assurance of phenotypic DST to its supranational reference laboratory (SRL), together with ethanol-preserved sputum specimens for LPA DST. Only 321 samples could be tested using LPA; of these, three were identified as being non-tuberculous mycobacteria using CTRL DST. Both tests had 269 sets with interpretable results. CTRL DST yielded almost the same number of interpretable results as LPA, with 90% concordance (κ = 0.612, P < 0.001). Five (1.9%) multidrug-resistant (MDR) strains, 46 (17.1%) resistant to INH only and 0 RMP only, were found by CTRL DST. For the LPA, these results were respectively 5 (1.9%), 26 (9.7%) and 2 (0.7%). With SRL DST as the gold standard, LPA was more accurate than CTRL DST for RMP, but missed almost half the INH-resistant samples. LPA applied directly on ethanol-preserved sputum specimens was similar to phenotypic DST in terms of yield of interpretable results. Although probably more accurate for RMP and MDR-TB, it appears to seriously underestimate INH resistance. Considering speed, easy and safe specimen transportation and low infrastructure requirements, LPA DST from sputum can be recommended for surveys in resource-poor settings.


A self-organized neural comparator.
Learning algorithms need generally the ability to compare several streams of information. Neural learning architectures hence need a unit, a comparator, able to compare several inputs encoding either internal or external information, for instance, predictions and sensory readings. Without the possibility of comparing the values of predictions to actual sensory inputs, reward evaluation and supervised learning would not be possible. Comparators are usually not implemented explicitly. Necessary comparisons are commonly performed by directly comparing the respective activities one-to-one. This implies that the characteristics of the two input streams (like size and encoding) must be provided at the time of designing the system. It is, however, plausible that biological comparators emerge from self-organizing, genetically encoded principles, which allow the system to adapt to the changes in the input and the organism. We propose an unsupervised neural circuitry, where the function of input comparison emerges via self-organization only from the interaction of the system with the respective inputs, without external influence or supervision. The proposed neural comparator adapts in an unsupervised form according to the correlations present in the input streams. The system consists of a multilayer feedforward neural network, which follows a local output minimization (anti-Hebbian) rule for adaptation of the synaptic weights. The local output minimization allows the circuit to autonomously acquire the capability of comparing the neural activities received from different neural populations, which may differ in population size and the neural encoding used. The comparator is able to compare objects never encountered before in the sensory input streams and evaluate a measure of their similarity even when differently encoded.


Prenatal diagnosis: current status and future trends.
Many of the major single-gene disorders in man can now be detected in utero using restriction fragment length polymorphisms (RFLPs). This approach is based on the knowledge of established linkage between RFLPs and mutations in an adjacent gene. The development of the polymerase chain reaction has altered the available approaches to this problem. This technique permits very rapid detection of the specific mutation from small amounts of starting material and may enable us to detect the genotype from fetal cells in maternal blood or from single cells in embryos before implantation. This may lead to the selection of embryos that lack a defect before they are implanted, avoiding the need for termination of pregnancy. Perhaps the greatest ethical challenge related to the application of these techniques will come with the understanding of the genetic basis for common polygenic disorders. The clarification of the multiple genetic factors responsible for a large part of susceptibility to diseases such as diabetes mellitus, rheumatoid arthritis or ischaemic heart disease will be greatly facilitated by a one-centimorgan genetic map. The strategy involved in establishing such a map, using the polymerase chain reaction, and then the identification of genetic loci responsible for susceptibility to a typical polygenic disease (insulin-dependent diabetes mellitus), is discussed. The ability to identify individuals at high risk of developing such diseases may present opportunities for early intervention.


Controlling the dominant magnetic relaxation mechanisms for magnetic hyperthermia in bimagnetic core-shell nanoparticles.
We report a simple and effective way to control the heat generation of a magnetic colloid under alternate magnetic fields by changing the shell composition of bimagnetic core-shell Fe3O4/ZnxCo1-xFe2O4 nanoparticles. The core-shell structure constitutes a magnetically-coupled biphase system, with an effective anisotropy that can be tuned by the substitution of Co2+ by Zn2+ ions in the shell. Magnetic hyperthermia experiments of nanoparticles dispersed in hexane and butter oil showed that the magnetic relaxation is dominated by Brown relaxation mechanism in samples with higher anisotropy (i.e., larger concentration of Co within the shell) yielding high specific power absorption values in low viscosity media as hexane. Increasing the Zn concentration of the shell, diminishes the magnetic anisotropy, which results in a change to a Néel relaxation that dominates the process when the nanoparticles are dispersed in a high-viscosity medium. We demonstrate that tuning the Zn contents at the shell of these exchange-coupled core/shell nanoparticles provides a way to control the magnetic anisotropy without loss of saturation magnetization. This ability is an essential prerequisite for most biomedical applications, where high viscosities and capturing mechanisms are present.


Sumoylation of Hes6 Regulates Protein Degradation and Hes1-Mediated Transcription.
Hes6 is a transcriptional regulator that induces transcriptional activation by binding to transcription repressor Hes1 and suppressing its activity. Hes6 is controlled by the ubiquitin-proteosome-mediated degradation system. Here we investigated the sumoylation of Hes6 and its functional role in its rhythmic expression. Hes6, SUMO, and ubiquitin were transfected into HeLa cells and the expression pattern was observed by Western blot and immunoprecipitation. To confirm the effect of sumoylation on the rhythmic expression of Hes6, we generated mouse Hes6 promoter-driven GFP-Hes6 fusion constructs and expressed these constructs in NIH 3T3 cells. Overexpression of SUMO led to sumoylation of Hes6 at both lysine 27 and 30. Protein stability of Hes6 was decreased by sumoylation. Moreover, expression of a Hes6 sumoylation-defective mutant, the 2KR (K27/30R) mutant, or co-expression of SUMO protease SUSP1 with native Hes6, strongly reduced ubiquitination. In addition, sumoylation was associated with both the rhythmic expression and transcriptional regulation of Hes6. Wild type Hes6 showed oscillatory expression with about 2-hour periodicity, whereas the 2KR mutant displayed a longer period. Furthermore, sumoylation of Hes6 derepressed Hes1-induced transcriptional repression. Hes6 sumoylation plays an important role in the regulation of its stability and Hes1-mediated transcription. These results suggest that sumoylation may be crucial for rhythmic expression of Hes6 and downstream target genes.